Expression Analysis And Function Identification Of CpEXP1Gene From Wintersweet (Chimonanthus Praecox (Linn.) Link)

Wintersweet (Chimonanthus praecox (Linn.) Link), Calycanthaceae, Chimonanthus Lindl., originated from China, is a precious woody cut for its important characteristics, such as blooming in deep winter, having strong stress resistance and fragrance et al.. However, serious bud and flower abscission, especially in the time of postharvest and storage, is a direct impediment to the exploitation of wintersweet cut. Previous studies about wintersweet cut preservation are mainly focused on physiological and biochemical characteristic during postharvest period. And studies of bud abscission is presently lacking.In recent years, studies showed that the relationship between expansin protein and organ abscission was closely. In2009, CpEXP1gene was isolated from cDNA library of wintersweet (GeneBank accession no. JN700522). Sequence analysis and homology comparison showed that CpEXP1gene was a kind of α-expansin gene. So if CpEXP1gene was related to the bud abscission of wintersweet, we could explore the mechanism by regulating the expression of this gene and seek for new solution to inhibit the bud abscission.In this dissertation, prokaryotic expression and protein activity were conducted based on gene clone. The expression of CpEXP1gene in different tissues and flowering phases, and the relationship among abscission layer develpoment, expression of CpEXP1gene and expansin protein activity was analysised. Functions of CpEXP1gene were studied by means of construction of plant expression vector and transformation of A. thaliana. At last, clone and function analysis of CpEXP1promoter was researched. Main results in this research are as follows:1. Prokaryotic expression and activity analysis of CpEXP1recombinant proteinBased on construction of prokaryotic expression vector pET32a(+)-CpEXP1, recombinant protein was obtained from the expression strain BL21(DE3). Purified recombinant protein was obtained by MagneHisTM Protein Purification Kit. Protein activity test showed that the recombinant protein had activity in vitor and could promote extension of hypocotyl collected from soybean. Medium supplemented with recombinant expansin indicated that expansin had no effect on differentiation of D. Maculata. However, it could promote proliferation and rooting. With the increase of concentration, the proliferation and rooting raised significantly.2. Flowering phases dividing and break strength classification Buds were borne on the first-order branches of the main stem mainly. And80.0%of the first-order branches were medium branch and spurs which were shorter than20cm. However the bud amounts on medium branches and spurses were up to82.4%. These medium branches and spurses contributed to the main ornamental value of wintersweet cut. Based on flower size, androgynophore, fragrance and so on, seven flowering phases were divided as follows:small-bud period, middle-bud period, big-bud period, early-bloom period, full-bloom period, early-wilt period and wilt period. Single-factor experiment showed that stem diameter was the factor most nearly associated with break strength. The correlation R2of stem diameter was0.1216, greater than bud diameter and stalk diameter, which’s correlation R2were0.0409,0.0369respectively. Based on break strength, buds in the middle-bud period were divided into four groups as follows:group Ⅰ:0N<break strength<1.4N, group Ⅱ:1.4<break strength<2.8N, group Ⅲ:2.8<break strength<4.2N, group IV:break strength>4.2N。3. Expression of CpEXPl gene, expansin activity and abscission layer development in wintersweetTo investigate the expression patterns of CpEXP1gene, Real-time PCR was employed to examine the expression levels in different tissues and flowering phases. Results showed that the expression of CpEXP1gene was temporal and spatial specific. The expression level was higher in floral organs than in vegetative organs. The order of CpEXP1gene expression from high to low was stamen, inner petal, pistil, full flower, middle petal, external petal, mature leaf, root, stem, cotyledon, young leaf. In analysis of seven flowering phases, RNA was extracted successfully except wilt period. Expression analysis showed significant differences in different phase. The order of CpEXP1gene expression from high to low was early-bloom period, full-bloom period, big-bud period, middle-bud period, early-wilt period, small-bud period. Under different break strength, the order of CpEXP1gene expression in pedicel from high to low was A (0N<break strength<1.4N), D(break strength≥4.2N),C (2.8<break strength<4.2N),B (1.4<break strength<2.8N). By measuring the expansin activity extracted from pedicel under different break strength, we found that the expansin activity was positive correlated with the expression of CpEXP1gene.To better understand the relationship among break strength, expansion protein activity and abscission layer development in wintersweet pedicel during the bud abscission period, break strength and expansion protein activity had been studied by dynamometer and protein recombinant in vitro respectively. Meanwhile, abscission layer formation had been observed based on paraffin section. Results showed that there was a negative correlation between break strength and the number of bud abscission and a positive correlation between break strength and abscission layer growth. The break strength could be seen as the basis of abscission stage division. In the course of bud abscission, expression of CpEXP1gene and expansin activity played an important role at the later stage. At the early stage of vase-holding, the expression of CpEXP1gene and expansin activity remained in lower level. The break strength declination and abscission layer development were relatively slow during this period. In later stage, the expression of CpEXP1gene and expansin activity increased rapidly accompanied by a significant decrease of break strength and serious cell disintegration in the abscission zone.4. Construction of plant expression vector of CpEXPl gene and transformation of A. thalianaBased on the nuclectide sequence submitted on the NCBI(GenBank accession number: JN700522), CpEXP1gene was cloned by reverse transcription PCR(RT-PCR) from the first strand of cDNA of Ch. praecox var. concolor Makino. In order to reveal the preliminary function of CpEXPl gene, plant expression vector’CpEXP1-2301G’ was constructed. Using inflorescence infiltration procedure mediated by Agrobacterium tumefaciens GV3101, transgenic T2plants were obtained. Compared with wild type, the length of inflorescence and leaf, leaf index and epidermal hair of transgenic plants increased significantly. The leaf reflexed seriously in transgenic A. thaliana and its withering period of leaf, the florescence and fructescence were in asvance distinctly. The break strength of mature fruit stalk in trans-plant was lawer than wild type. All these indicated that the expression of CpEXP1gene played an important role in the course of morphogenesis, growth cycle and abscission.5. Isolation, bioinformatics and expression analysis of wintersweet CpEXPl promoterPromoter sequence of CpEXP1genes (named CpEXP1-pro) which including2521bp was isolated from the genomic DNA of wintersweet by hiTAIL-PCR. Sequence analysis suggested that the sequence had a series of elements that related to photoresponses, high or low temperature sresee, salicylic acid and specific expression in seeds and endosperm. Using agrobacterium-mediated transformation, PBI121-CpEXP1-pr was transfered into tobacco for transient expression and into A. thaliana for stable expression. Results showed that CpEXP1-pr could drive the expression of GUS gene in tobacco leafe. In transgenic A. thaliana, the expression of GUS gene was temporal and spatial specific. The expression level was higher in seeds at the stage of germination and plant at the stage of flowering and seeding while no expression was detected in seedings with two cotyledon and seedings at euphylla stage. With the maturation and senescence of A. thaliana leaves, the expression of GUS gene increased gradually, especially at the basal of petiole. During the period of infruit growth, the expression decreased and no expression was detected in mature infruit except in fruit stalk. We speculated that CpEXP1-pro was closely related to germination, fruit expansion and abscission of leaf and fruit.Combined with results of Real-time PCR, expression analysis of CpEXP1gene in wintersweet and A. thaliana, promoter analysis, conclutions were as follow:the expression of CpEXPl gene was temporal and spatial specific. It played an important role in the course of morphogenesis, growth cycle and abscission.